The RNA isolated from the agarose beads at the “Elute” step of the RNA immunoprecipitation (RIP) assay was used for the amplification and the RACE assays (SMARTer RACE kit; Clontech, Takara Bio Inc., Kusatsu, Japan) to determine the 5′ and 3′ ends of co-immunoprecipitated RNA. To determine the 5′ end, a random primer and manufacturer-supplied adapter oligos were used for the first-strand cDNA synthesis. The 5′ RACE product was amplified using the forward primer supplied by the manufacturer and a CTV-specific reverse primer (GSP1). To determine the 3′ end of the RNA, the isolated RNA was polyadenylated using Escherichia coli poly (A) polymerase (Applied Biosystems, Waltham, MA, USA). The first strand synthesis and 3′ RACE was performed as instructed (SMARTer RACE kit; Clontech, Takara Bio Inc., Kusatsu, Japan) with a manufacturer-supplied reverse primer and a CTV-specific forward primer (GSP2). Both 5′ and 3′ RACE products were cloned into the manufacturer-supplied vector and selected clones (10 for 5′ RACE and 100 for 3′ RACE) were sequenced (Psomagen, Rockville, MD, USA).
Smarter race kit
Manufactured by Takara Bio
Sourced in United States, Japan, China
The SMARTer RACE kit is a molecular biology tool designed for rapid amplification of cDNA ends (RACE). It enables efficient and unbiased amplification of 5' and 3' cDNA ends from small amounts of total RNA or poly(A)+ RNA.
Automatically generated - may contain errors
Lab products found in correlation
Pmd18 t vector, by Takara Bio (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Pgem t easy vector system, by Promega (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Peasy t1 vector, by Transgene (2 mentions)
Pgem t easy vector, by Promega (2 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (2 mentions)
One shot max efficiency dh5α t1 competent cells, by Thermo Fisher Scientific (2 mentions)
Advantage 2 pcr kit, by Takara Bio (2 mentions)
μmacs oligo dt microbeads, by Miltenyi Biotec (2 mentions)
Nucleospin extract 2 gel extraction kit, by Macherey-Nagel (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Superscript 2, by Thermo Fisher Scientific (2 mentions)
Facsaria 3, by BD (2 mentions)
Oligo dt primer, by Promega (1 mentions)
Rq1 rnase free dnase 1, by Promega (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Improm 2 reverse transcriptase, by Promega (1 mentions)
Pgm t vector, by Tiangen Biotech (1 mentions)
59 protocols using smarter race kit
1
Determining RNA 5' and 3' Ends by RACE
2
RACE and cloning of sheep ACSL1 gene
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Primers were designed according to the principles of rapid amplification of cDNA ends (RACE) primer design using ACSL1 gene sequences from GenBank. Primer Premier 6.0 software was used to design 3′UTR primers and labeled as SP1, SP2, SP3, and SP4 (Table 1 ).
Reverse transcription, 5′-end phosphorylation of primers, and ligation assays were performed according to modified protocols of the manufacturers of SMARTer RACE Kit (Clontech). Tissue RNA was extracted and reverse transcribed with specific primers followed by nested PCR with the primers listed inTable 1 .
The target fragment was amplified according to the RACE system. PCR reaction cycles are as follows: 94°C for 30 s, 72°C for 3 min, five cycles; 94°C for 30 s, 70°C for 30 s, 72°C for 3 min, five cycles; 94°C for 30 s, 68°C for 30 s, 72°C for 3 min, 25 cycles. After ligating with pmd18-t vector, the fragment was sequenced by a biological company.
Based on the RACE result, primers F1, R1, F2, and R2 (Table 1 ) were used to clone the CDS regions of two different transcripts of the sheep ACSL1 gene. ACSL1-a was amplified with primers F1 and R1, while amplification of ACSL1-b was carried out with F2 and R2. A biological company sequenced the target fragment that had been ligated with the pmd18-t vector.
Reverse transcription, 5′-end phosphorylation of primers, and ligation assays were performed according to modified protocols of the manufacturers of SMARTer RACE Kit (Clontech). Tissue RNA was extracted and reverse transcribed with specific primers followed by nested PCR with the primers listed in
The target fragment was amplified according to the RACE system. PCR reaction cycles are as follows: 94°C for 30 s, 72°C for 3 min, five cycles; 94°C for 30 s, 70°C for 30 s, 72°C for 3 min, five cycles; 94°C for 30 s, 68°C for 30 s, 72°C for 3 min, 25 cycles. After ligating with pmd18-t vector, the fragment was sequenced by a biological company.
Based on the RACE result, primers F1, R1, F2, and R2 (
3
FincoR Transcription Mapping by RACE
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5’ and 3’ RACE assays were performed using a SMARTer RACE kit (Clontech) according to the manufacturer’s instructions. The resulting PCR products were separated by electrophoresis in agarose gels and cloned into the pRACE vector provided by the kit. The transcription start sites and end sites of FincoR were determined by sequencing. The gene-specific primers used for 5’- and 3’-RACE are listed in Supplementary Table S6 .
4
Identifying PcmiR397 Target Genes in Pear
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Computational prediction of PcmiR397 target genes was conducted by local BLASTn search as previously reported (Allen et al., 2005 (link); Liu et al., 2015 (link); Schwab et al., 2005 (link)). The cleavage site in the PcLAC genes mediated by PcmiR397 was validated by RNA ligase‐mediated (RLM)‐5′‐RACE (Llave et al., 2011 ). 5′ DNA adaptor was synthesized by the Tianyihuiyuan Biotech Company and this was ligated to 3 μg of total RNA from Conference pear in vitro plantlets. cDNA was synthesized from the verified ligated RNA as template using oligo(dT) primers. Cleavage products of target genes were amplified by nested PCR using 5′ adaptors and 3′ gene‐specific nested primers. In addition, cleavage site verification of PpLACs in Jinshui No. 2 was performed using a Smarter RACE Kit (Clontech). Sequences of the 5′ adaptor and gene‐specific primers are listed in Table S6 . PCR products were ligated to the pMD18‐T vector (Takara) and 5–10 sequenced randomly selected clones were sequenced by the Tianyihuiyuan Biotech Company to identify and analyse the cleavage sites.
5
Cloning and Characterization of NF-κB Factors in M. sexta
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In the M. sexta EST library (http://entoplp.okstate.edu/profiles/jiang.htm ), two EST fragments were predicted to encode Rel-homology domain (RHD)-containing proteins (manduca.Contig2427 and manduca.Contig7025). Gene specific primers were designed based on the EST sequences to clone the full-length cDNAs. Total RNA was prepared from the fat body of day 3 M. sexta naïve larvae using TRIzol® Reagent (T9424, Sigma–Aldrich), and contaminated genomic DNA was removed by RQ1 RNase-free DNase I (Promega). Reverse transcription was performed using oligo(dT) primer (Promega) and ImProm-II reverse transcriptase (Promega) following the manufacturer’s instructions. The 5′ and 3′ RACE reactions were performed using smarter race kit (Clontech). The opening reading frame (ORF) was predicted from the nucleotide sequence using DNAMAN (Lynnon Corporation, Quebec, Canada). BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi ) was used to search homologous RHD sequences. RHD sequences from various NF-κB factors were aligned with the MUSCLE module of MEGA 6.0. The aligned sequences were used to construct a neighbor-joining tree with 1000 Bootstrap Replications61 (link). RHD sequences were aligned with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/ ), and the alignment result was decorated with ESPript 3.062 (link). Consensus sites of κB sites were displayed with WebLogo363 (link)64 (link).
6
Rapid Amplification of cDNA Ends
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The 5′- and 3′-rapid amplification of cDNA ends (RACE) experiments were performed using the SMARTer RACE Kit (Clontech) according to the instructions of the manufacturer. The RACE PCR products were separated on an agarose gel. PCR bands were cloned into the pGM-T vector (TIANGEN Biotech, China) and sequenced. The RACE primers targeting Gm18840 used for the nested PCR are listed below: 5′-RACE outer primer, 5′-CCATGTGCCATCATGCTGGCAAA-3′; 5′-RACE inner primer-1, 5′-CTGGTAGCCTTGCTTGCAGGTGACA-3′; 3′-RACE outer primer, 5′-CCTGCAAGCAAGGCTACCAGCTCAT-3′; and 3′-RACE inner primer, 5′-ATGCCCAGGTGCAAGAT CAAGAACTGTG-3′.
7
Whole Genome Amplification of Lyssavirus
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For the whole genome amplification of lyssavirus, twelve sets of RT-PCR primers were used [4 (link)], and a few of the primers were modified according to the sequences of the isolate (Table S1 ). The RT-PCRs were performed using the SuperScript III One-Step RT-PCR System with Platinum Taq polymerase High Fidelity kit (Invitrogen, Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instruction. The reaction was initiated at 42 °C for 40 min and then at 95 °C for 2 min, followed by 35 cycles of 95 °C for 40 s, 50 °C for 50 s, and 72 °C for 80 s, and the reaction ended after a final extension of 72 °C for 7 min.
The termini sequences at the 3′ and 5′ ends of viral genome were obtained using the SMARTer RACE kit (Clontech Laboratories, TaKaRa Bio Company, Mountain View, CA, USA) following the manufacturer’s instructions. Due to the fact that the amount of bat brain tissue was limited, the nucleic acid extracted from the cell culture supernatant of the virus isolation was used for the amplification of the termini sequences.
The RT-PCR products were sequenced with the 3700XL DNA analyzer (Applied Biosystems, Waltham, MA, USA) by a commercial sequencing service (Mission Biotech Co., Taipei, Taiwan).
The termini sequences at the 3′ and 5′ ends of viral genome were obtained using the SMARTer RACE kit (Clontech Laboratories, TaKaRa Bio Company, Mountain View, CA, USA) following the manufacturer’s instructions. Due to the fact that the amount of bat brain tissue was limited, the nucleic acid extracted from the cell culture supernatant of the virus isolation was used for the amplification of the termini sequences.
The RT-PCR products were sequenced with the 3700XL DNA analyzer (Applied Biosystems, Waltham, MA, USA) by a commercial sequencing service (Mission Biotech Co., Taipei, Taiwan).
8
Cloning Schistosoma mansoni 5HTR_L
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Sequence for Sm.5HTRL was determined by cloning from an Schistosoma mansoni cDNA library (adult male and female NMRI strain, BEI cat #NR-36056) using high fidelity Advantage HD DNA polymerase (Clontech) and primers described in [20 (link)]. PCR products were ligated into the pGEM-T Easy vector system (Promega) prior to DNA sequencing. Additional sequence contained in the Sm.5HTRL isoform was verified by 5’/3’ RACE (SMARTer RACE Kit, Clontech) using total RNA extracted from S. mansoni (Trizol reagent, Ambion). Products were cloned into the pRACE vector (In-Fusion HD Cloning Kit, Clontech) prior to DNA sequencing.
9
Sepiapterin Reductase Gene Cloning in White Shrimp
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The partial cDNA sequence of sepiapterin reductase was obtained from transcriptomic sequencing of L. vannamei by TBLASTN search of the NCBI database. The specific primer pair (LvSprF/R, Table 1 ) was designed to amplify the gene fragment from white shrimp hemocytes cDNA. In addition, 3’ and 5’ rapid amplification of cDNA ends (RACE) PCR was carried out using the SMARTer RACE kit (Clontech, Japan), following the user manual with the nested primers listed in Table 1 . The PCR products were purified using a Gel Extraction Kit (Tiangen, China) and cloned into the pMD-18T vector (TaKaRa). The plasmids were transformed into competent E. coli DH5α cells, and positive colonies were screened by colony PCR and then sequenced. The full-length LvSpr cDNA was obtained by assembling the overlapping sequences.
10
Cloning and Sequencing of TM7SF2 cDNA
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The TM7SF2 cDNA was cloned from HeLa cells by RT-PCR. Total RNAs were isolated from HeLa cells cultured in cholesterol-restrictive medium and reversed transcribed into cDNA using a cDNA amplification kit (SMARTer RACE kit, Clontech). The TM7SF2 cDNA was amplified using a universal forward primer (5’-CTAATACGACTCACTATAGGGC-3’) and a gene-specific reverse primer (5’-TCAGTAGATGTAGGGCATGATGCG-3’). The resulting PCR product was subjected to Sanger sequencing.
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